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In-depth investigation of the species problem and taxonomic status of marbled crayfish, the first asexual decapod crustacean

The marbled crayfish is the only obligately parthenogenetic decapod crustacean and a novel research model and invasive animal on three continents. It is regarded either as a parthenogenetic form of slough crayfish Procambarus fallax or as a separate species named Procambarus virginalis. In order to investigate the species question of this unusual crayfish in detail we have identified the similarities and differences in morphology, life history, genetics, behaviour, ecology and biogeography between marbled crayfish and its most likely parent species P. fallax. We have investigated specimens from natural habitats, laboratory colonies and museum collections and performed a meta-analysis of our data and published data. Our COI based molecular tree with 27 Cambaridae confirms closest relationship of marbled crayfish with P. fallax. Marbled crayfish and P. fallax are similar with respect to morphological characters, coloration and body proportions, but differ considerably with respect to body size, fertility and longevity. The mitochondrial genes of both crayfish are similar, but ploidy level and haploid genome size are markedly different. Both crayfish are eurytopic and have two major annual recruitment periods, but marbled crayfish show different population structure and higher invasiveness. Marbled crayfish occur in tropical to cold temperate habitats of the old world, but P. fallax is confined to subtropical and warm-temperate habitats of the southeastern USA. Cross-breeding experiments with both crayfish revealed reproductive isolation. The application of the Evolutionary Genetic Species Concept for asexuals to all available data supports raising marbled crayfish from \"forma\" to species rank. A determination key is provided to discriminate Procambarus virginalis, the first asexual decapod species, from its parent species P. fallax.

zoology

The return to water in ancestral Xenopus was accompanied by a novel mechanisms for producing and shaping vocal signals

Species-specific vocal signals allow listeners to locate potential mates. During the tetrapod transition from water to land, lungs replaced gills, allowing expiration to drive sound production. Several groups, e.g. cetaceans and some frogs, then returned to water. Here we explore how air-driven sound production changed upon re-entry and how essential acoustic information on species identity was preserved in the secondarily aquatic frog Xenopus. We filmed movements of cartilage and muscles during evoked sound production in isolated larynges. Our results refute the current theory for Xenopus vocalization, cavitation, and instead favor sound production by mechanical excitation of laryngeal resonance modes following rapid separation of laryngeal arytenoid discs. The resulting frequency resonance modes (dyads) are intrinsic to the larynx rather than due to neuromuscular control. We show that dyads are a distinctive acoustic signature across species. While dyad component frequencies overlap across species, their ratio is shared within each Xenopus clade and thus provide information on species identity, potentially facilitating both conspecific localization and ancient species divergence.

zoology

Latitudinal Variation in Life History Reveals a Reproductive Disadvantage in the Texas Horned Lizard (Phrynosoma cornutum)

Geographically widespread species that occupy many thermal environments provide testable models for understanding the evolution of life-history responses to latitude, yet studies that draw range-wide conclusions using descriptive data from populations in the core of a species distribution can overlook meaningful inter-population variation. The phrynosomatid lizard Phrynosoma cornutum spans an extensive latitudinal distribution in North America and has been well-studied in connection with life-history evolution, yet populations occupying the most northern and coldest areas within its range were absent from previous examinations. We tested genus-wide models and challenged species-specific findings on the evolution of the life-history strategy for P. cornutum using populations at the northern edge of its geographic range and comparative material from farther south. Multivariate analyses revealed that egg dimensions decreased with clutch size, suggestive of a previously unrecognized tradeoff between egg size and egg number in this species. Interestingly, reproductive traits of females with shelled eggs did not covary with latitude, yet we found that populations at the highest latitudes typified several traits of the genus and for the species, including a model for Phrynosoma of large clutches and delayed reproduction. A significant deviation from earlier findings is that we detected latitudinal variation in clutch size. This finding, although novel, is unsurprising given the smaller body sizes from northern populations and the positive relationship between clutch size and body size. Intriguing, however, was that the significant reduction in clutch size persisted when female body size was held constant, indicating a reproductive disadvantage to living at higher latitudes. We discuss the possible selective pressures that may have resulted in the diminishing returns on reproductive output at higher latitudes. Our findings highlight the type of insights in the study of life-history evolution that can be gained across Phrynosomatidae from the inclusion of populations representing latitudinal endpoints.

zoology

Life history of the Rio Grande leopard frog (Lithobates berlandieri) in Texas

We ascertained various life-history traits from an examination of 310 museum specimens of the Rio Grande leopard frog (Lithobates berlandieri Baird, 1859) collected during 1907-2016 from Texas, USA. Lithobates berlandieri was captured during every month of the year except November, and adults were most frequently encountered during January-September with a distinct peak in May. Mean body size of adult males (69.5 mm) was smaller than that of adult females (77.5 mm), and both sexes were larger in mean body sizes than those of New Mexico populations (M = 64.4 mm; F = 73.5 mm). Females were gravid during January-September, and most gravid females were captured from late-winter to early-summer. Gonadal enlargement in males was generally high throughout January-September with no detectable seasonal increase. Feeding became widespread in both sexes during May-June shortly after a spring breeding bout. Spent females were common in July and lipid deposition increased in June/July, signaling oogenesis for breeding in the fall. From 15 gravid females, we estimated a mean clutch size of 3,107 eggs which was correlated with female body size, yet egg diameter was not related to clutch or body size. Age to metamorphosis was likely 2 to 4 months depending upon whether eggs were laid in the winter/spring or late fall. If metamorphosis occurred in May/June, the minimum size at sexual maturity in adult males (50.1 mm) could have been reached in 3-4 months and in 6-7 months for adult females (57.2 mm). Mean adult body sizes, however, may have taken 12 to 17 months to reach. A synthesis across Texas populations suggests that the breeding season extends almost continuously from the fall through the winter and spring until mid-summer and is interrupted by winter and summer peaks in seasonal temperatures.

zoology

Measuring Orangutan nest structure using Unmanned Aerial Vehicle (UAV) and ImageJ

The nest is one of the crucial elements in orangutan daily activities. Previously, most of the nest structure studies were done manually by estimating measurement directly from visual observation. However, using the latest unmanned aerial vehicle (UAV) technology, we can reduce the workforce, time and energy while simultaneously ensuring the safety of the researcher conducting nest structure analysis. We recorded 49 pictures of orangutan nests at Sepilok Orangutan Rehabilitation Centre (SORC) using UAV (DJI Phantom 3 Quadcopter). The nest structure (length, depth, and width) was digitally measured by using ImageJ. Most of the nests were built at a strong, stable, and comfortable position at the top of the tree. Most orangutans chose Eusideroxylon zwageri to build nest compared to other tree species because of the strong and durable wood characteristic which would create a sturdy, strong and comfortable nest. We propose the use of drone with digital image analysis could provide a more accurate, less time consuming and safe method for studying orangutan nest structure.

zoology

qPCR assays for the detection of two western North American freshwater mussel species, Anodonta nuttalliana and Anodonta oregonensis, from environmental DNA

We developed species-specific quantitative PCR assays for the detection of two freshwater mussel species native to the western North America, Anodonta nuttalliana and Anodonta oregonensis, from environmental DNA. These species have experienced dramatic declines over the last century, and are currently threatened in many portions of their range. Improved tools for detecting and monitoring these species are needed. Species-specificity and sensitivity of the assays was empirically tested in the lab, and both assays were also validated with field collected eDNA samples. We found that the assays we designed are species-specific, sensitive, and are effective for detecting Anodonta nuttalliana and Anodonta oregonensis from environmental DNA samples collected from streams and rivers. These assays will aid in the detection, monitoring, management, and conservation of these threatened species.

zoology

The Effect of Phylogenetic Uncertainty and Imputation on EDGE Scores

Faced with the challenge of saving as much diversity as possible given financial and time constraints, conservation biologists are increasingly prioritizing species on the basis of their overall contribution to evolutionary diversity. Metrics such as EDGE (Evolutionary Distinct and Globally Endangered) have been used to set such evolutionarily-based conservation priorities for a number of taxa, such as mammals, birds, corals, amphibians, and sharks. Each application of EDGE has required some form of correction to account for species whose position within the tree of life are unknown. Perhaps the most advanced of these corrections is phylogenetic imputation, but to date there has been no systematic assessment of both the sensitivity of EDGE scores to a phylogeny missing species, and the impact of using imputation to correct for species missing from the tree. Here we perform such an assessment, by simulating phylogenies, removing some species to make the phylogeny incomplete, imputating the position of those species, and measuring (1) how robust ED scores are for the species that are not removed and (2) how accurate the ED scores are for those removed and then imputed. We find that the EDGE ranking for species on a tree is remarkably robust to missing species from that tree, but that phylogenetic imputation for missing species, while unbiased, does not accurately reconstruct species evolutionary distinctiveness. On the basis of these results, we provide clear guidance for EDGE scoring in the face of phylogenetic uncertainty.

zoology

Social and nutritional factors shape larval aggregation, foraging, and body mass in a polyphagous fly

The majority of insect species have a clearly defined larval stage during development. Larval nutrition is crucial for individuals growth and development, and larval foraging success often depends on both resource availability and competition for those resources. To date, however, little is known about how these factors interact to shape larval development and behaviour. Here we manipulated the density of larvae of the polyphagous fruit fly pest Bactrocera tryoni ( Queensland fruit fly), and the diet concentration of patches in a foraging arena to address this gap. Using advanced statistical methods of machine learning and linear regression models, we showed that high larval density results in increased larval aggregation across all diets except in extreme diet dilutions. Larval aggregation was positively associated with larval body mass across all diet concentrations except in extreme diet dilutions where this relationship was reversed. Larvae in low-density arenas also tended to aggregate while those in high-density arenas tended to disperse, an effect that was observed for all diet concentrations. Furthermore, larvae in high-density arenas displayed significant avoidance of low concentration diets - a behaviour that was not observed amongst larvae in low-density arenas. Thus, aggregation can help, rather than hinder, larval growth in high-density environments, and larvae may be better able to explore available nutrition when at high-density than when at low density.

zoology

Where do animals covered by the US Animal Welfare Act live? Analysis of USDA licensed and registered entities

Nonhuman animals are housed in captivity for a range of purposes in the US and other countries. The regulation, oversight, and public transparency of decisions related to the care and use of those animals varies by species and activity. The goals of this paper are 1) to provide a concise and accessible summary of the number and types of facilities either registered or licensed by the USDA and overseen by the federal agency; and 2) to provide concise comparisons of the relative proportion of different types of use of nonhuman animals that fall under the AWA (Animal Welfare Act). Analysis of the publicly-available list of USDA certificate holders produced descriptive data for each state and the US overall. Licensed exhibitors (N=2,640; 33% of total) and breeders (N=2,701; 34% of total) comprise two-thirds of the 8,002 USDA certificate holders. Registered research facilities (N=1,110) account for 14% of the total USDA certificates. The final 19% consists of licensed dealers (N=763; 9%) and registered carriers or interim handlers (N=788; 10%). The number and distribution of types of certificates varies across states. The largest number of exhibitors and dealers are in Florida, while the largest number of breeders are in Missouri. California has the largest number of research registered facilities. Finally, a comparison of the estimated number of animals in the US that are pets, used in agriculture, exhibition, and research suggests that animals in research are between 0.07-2.3% of the total. The comparison also highlights areas in which public information about captive animals in the US is uneven or missing altogether. Together, the current report provides a concise view of basic information and data relevant to public consideration and policy decisions about animals housed in the set of captive settings to which the AWA applies.

zoology

A Taxonomic Review and Revisions of Microstomidae (Platyhelminthes: Macrostomorpha)

Microstomidae (Platyhelminthes: Macrostomorpha) diversity has been almost entirely ignored within recent years, likely due to inconsistent and often old taxonomic literature and a general rarity of sexually mature collected specimens. Herein, we reconstruct the phylogenetic relationships of the group using both previously published and new 18S and CO1 gene sequences. We present some taxonomic revisions of Microstomidae and further describe 8 new species of Microstomum based on both molecular and morphological evidence. Finally, we briefly review the morphological taxonomy of each species and provide a key to aid in future research and identification that is not dependent on reproductive morphology. Our goal is to clarify the taxonomy and facilitate future research into an otherwise very understudied group of tiny (but important) flatworms.

zoology

Moving in complex environments: a biomechanical analysis of locomotion on inclined and narrow substrates

Characterisation of an organisms performance in different habitats provides insight into the conditions that allow it to survive and reproduce. In recent years, Northern quolls (Dasyurus hallucatus)--a medium-sized semi-arboreal marsupial native to northern Australia--have undergone significant population declines within open forest, woodland and riparian habitats, but less so in rocky areas. To explore this decline, we quantified the biomechanical performance of wild Northern quolls over inclined narrow (13 mm pole) and inclined wide (90 mm platform) substrates. We predicted that quolls may possess biomechanical adaptations to increase stability on narrow surfaces, which are more common in rocky habitats. Our results display that quolls have some biomechanical characteristics consistent with a stability advantage on narrow surfaces. This includes the coupled use of limb pairs and high grip torques (Max = 68.9 N.m, Min = -69.9 N.m), highlighting their ability to generate corrective forces to counteract the toppling moments commonly encountered during gait on narrow surfaces. However, unlike other arboreal specialists such as primates, speed was constrained on narrow surfaces, and quolls did not adopt diagonal sequence gaits. Quolls appear to adopt similar performance characteristics to cats and dogs which may limit their ability to outperform these key predators (invasive or otherwise) in particular habitats. This overlap may contribute to the declining population of Northern quolls on mainland Australia.

zoology

Reducing Aedes albopictus breeding sites through education: a study in urban area

Aedes albopictus tends to proliferate in small, often man-made bodies of water, largely present in urban private areas. For this reason, education and community participation are considered crucial for source reduction and mosquito control. In the current study, we tried to relate for the first time in Greece, the effectiveness of resident education in an urban area with the number of breeding sites based on previous KAP (knowledge, attitudes and practices) studies. Our study examines the relationship between mosquito breeding sources and socioeconomic or demographic characteristics of different households in a Greek municipality and evaluates efficacy of resident education. The results revealed that only a minority of residents knew where mosquitoes breed (18.6%) and only 46% felt that residents had any responsibility for managing breeding habitat. Our findings strongly suggest that only the presence of scientific staff inspecting possible habitats in their properties, could be enough to stimulate practices towards source reduction. However, educational interventions alone with printed education material cannot enhance significant community participation and source reduction.

zoology

Delayed start of first-time breeding and non-breeders surplus in the Western Siberian population of the European Pied Flycatcher

Non-breeders are those sexually mature individuals that do not breed in a given reproductive cycle of a population. There is a widespread belief that the presence of non-breeders can affect the actual population dynamics, as well as the population responses to environmental change (Lee et al. 2017). Sternberg (1989), using demographic data, has shown that 83% and 62% of males and females, respectively, do not breed in the first year of life in the Lower Saxony (Germany) population of the European Pied Flycatcher (Ficedula hypoleuca). Later, with experimental removal of males, it has been proven that in the Lower Saxony and Moscow Region (Russia) populations, there are many non-breeding males (Sternberg et al. 2002). For the Netherlands population of the European Pied Flycatcher, the presence of a large number of non-breeders has been demonstrated using experimental removals for both males and females (Both et al. 2017). Here we have estimated the number of non-breeders in the Western Siberian population of the European Pied Flycatcher using demographic data (11 cohorts from 2001 to 2011 of birth) and experimental removal of males. We have shown that both males and females can start to breed at the age of one to five years. The proportion of non-breeders can be 59.5% and 68.5% for first-year males and females, respectively. We discuss the differences in the proportion of non-breeders between the Western Siberian and European populations of the European Pied Flycatcher, as well as factors affecting the number of non-breeders.

zoology

Influence of temperature on the development, reproduction and regeneration in the flatworm model organism Macrostomum lignano

The free-living marine flatworm Macrostomum lignano is a powerful model organism to study mechanisms of regeneration and stem cell regulation due to its convenient combination of biological and experimental properties, including the availability of transgenesis methods, which is unique among flatworm models. However, due to its relatively recent introduction in research, there are still many biological aspects of the animal that are not known. One of such questions is the influence of the culturing temperature on Macrostomum biology. Here we systematically investigated how different culturing temperatures affect the development time, reproduction rate, regeneration, heat shock response, and gene knockdown efficiency by RNA interference in M. lignano. We used marker transgenic lines of the flatworm to accurately measure the regeneration endpoint and to establish the stress response threshold for temperature shock. We found that compared to the culturing temperature of 20{degrees}C commonly used for M. lignano, elevated temperatures of 25{degrees}C-30{degrees}C substantially speed-up the development and regeneration time and increase reproduction rate without detectable negative consequences for the animal, while temperatures above 30{degrees}C elicit a heat shock response.\n\nWe show that altering the temperature conditions can be used to shorten the time required to establish M. lignano cultures, store important lines and optimize the microinjection procedures for transgenesis. Our findings will help to optimize the design of experiments in M. lignano and thus facilitate future research in this model organism.

zoology

The comparative and genetic methods for East European Unionidae taxonomy

Taxonomy of freshwater mussels from family Unionidae has been ambiguous for a long time. A number of methods used for their identification, including the so-called comparative method, are based on shell morphology. However, this morphology turned out to have a high level of within-species variation, and the shape of the shell of a specimen depends strongly on its environment and conditions of growth. For these reasons, the number of species recognized by the comparative method kept increasing. We applied both the comparative morphological method and methods of molecular genetics to address the taxonomy of Unionidae. We performed the comprehensive study of 70 specimens of Unionidae mussels collected from the River Ivitza, Volga basin. The specimens represented 14 comparative species, belonging to 4 comparative genera of Unionidae: Colletopterum, Pseudanodonta, Unio and Crassiana. Sequencing of the nuclear (ITS1) and mitochondrial (COI, 16S rDNA) genetic regions revealed 5 groups with high within-group genetic homogeneity separated from each other by long genetic distances. According to the comparison with the available sequences, these groups correspond to 3 Eastern European genera and 5 species: Anodonta anatina, Pseudanodonta complanata, Unio pictorum, Unio tumidus and Unio crassus. The results obtained indicate that the comparative method is inappropriate for the taxonomic analysis of East European Unionidae.

zoology

Co-invasion of the ladybird Harmonia axyridis and its parasites Hesperomyces virescens fungus and Parasitylenchus bifurcatus nematode to the Caucasus

Study of parasites in recently established populations of invasive species can shed lite on sources of invasion and possible indirect interactions of the alien species with native ones. We studied parasites of the global invader Harmonia axyridis (Coleoptera: Coccinellidae) in the Caucasus. In 2012 the first established population of H. axyridis was recorded in the Caucasus in Sochi (south of European Russia, Black sea coast). By 2018 the ladybird has spread to the vast territory: Armenia, Georgia and south Russia: Adygea, Krasnodar territory, Stavropol territory, Dagestan, Kabardino-Balkaria and North Ossetia. Examination of 213 adults collected in Sochi in 2018 have shown that 53% of them are infested with Hesperomyces virescens fungi (Ascomycota: Laboulbeniales) and 8% with Parasitylenchus bifurcatus nematodes (Nematoda: Tylenchida, Allantonematidae). Examined H. axyridis specimens were free of parasitic mite Coccipolipus hippodamiae. An analysis of the phylogenetic relationships of Parasitylenchus bifurcatus based on 18S rDNA confirmed the morphological identification of this species. Hesperomyces virescens and Parasitylenchus bifurcatus are firstly recorded from the Caucasus and Russia, though widespread in Europe. It probably indicates that they appeared as a result of co-invasion with their host. Harmonia axyridis was released in the region for pest control, but laboratory cultures are always free of H. virescens and P. bifurcatus. Therefore, detection of H. virescens and P. bifurcatus indicates that population of H. axyridis in the Caucasus cannot derive exclusively from released specimens. We did not find H. virescens on 400 specimens of 31 other ladybird species collected in the same localities with H. axyridis in the Caucasus. No reliable correlation between infestation by H. virescens and P. bifurcatus has been found. Besides these two parasites an unidentified species of the order Mermithida is recorded. It is the first documented case of H. axyridis infestation by a parasitic nematode of this order in nature.

zoology

MicroRNAs are likely part of the molecular toolkit regulating adult reproductive diapause in the mosquito, Culex pipiens

For many arthropods, including insects, diapause is the primary mechanism for survival during unfavorable seasons. Although the exogenous signals and endogenous hormones that induce and regulate diapause are well-characterized, we still lack a mechanistic understanding of how environmental information is translated into molecular regulators of the diapause pathway. However, short, non-coding microRNAs (miRNAs) are likely involved in generating both the arrested egg follicle development and fat hypertrophy in diapausing females of the Northern house mosquito, Culex pipiens. To determine whether miRNAs might respond to changes in day length and/or regulate diapause pathways, we measured the abundance of candidate miRNAs in diapausing and nondiapausing females of Cx. pipiens across the adult lifespan. Of the selected miRNAs nearly all were more abundant in nondiapausing females relative to diapausing females, but at different times. Specifically, miR-13b-3p, miR-14-3p, miR-277-3p, and miR-305-5p were upregulated in nondiapausing females early in adulthood, while miR-309-3p and miR-375-3p were upregulated later in adult life, and miR-8-3p and miR-275-3p were upregulated both early and late in adult life. Taken together, our data demonstrate that miRNA expression is dynamic, changing across adult lifespan. Further, differential miRNA expression between diapausing and nondiapausing females of Cx. pipiens suggests that this epigenetic mechanism is part of the molecular toolkit regulating diapause.

zoology